Novel Melanoma Antigen Peptide and Uses Thereof

ABSTRACT

The present invention relates to novel melanoma antigen peptides and specific T lymphocytes directed to said peptides and the use thereof for treating melanoma.

FIELD OF THE INVENTION

The invention relates to melanoma antigen peptides named MELOE and theiruse for preventing, treating and diagnosing melanoma.

BACKGROUND OF THE INVENTION

Melanomas are aggressive, frequently metastatic tumors derived fromeither melanocytes or melanocyte related nevus cells. Melanomasrepresent approximately three percent of all skin cancers and even whenit is apparently localized to the skin, up to 30% of the patients willdevelop systemic metastasis. Classic modalities of treating melanomainclude surgery, radiation and chemotherapy. Then immunotherapy and genetherapy have emerged as promising methods for treating melanoma.Rosenberg's results showed that adoptive transfer into patients withmetastatic melanoma of tumor infiltrating lymphocytes (TIL) thatrecognize cancer antigens, are able to mediate the regression ofmetastatic cancer in 35 to 40% of melanoma patients.

In the last twenty years, many human melanoma antigens recognized by Tcells have been identified using various methods such as cDNA cloning,MHC-bound peptide purification or T cell induction against candidatepeptides or proteins. These antigens have been classified in severalgroups: melanocytic differentiation antigens (such as Melan-A/MART-1)(1), cancer-germline antigens, shared by several tumors and malegermline cells (such as MAGE antigens) (2, 3), mutated antigensgenerated by genetic alterations (such as CDK4) (4), antigensoverexpressed in various tumor types (such as PRAME) (5), and antigensaberrantly expressed in tumors (such as NA17-A and NA88-A) (6, 7).However, despite their high number, the immunogenicity of these antigenshas not been elucidated yet, with the exception of Melan-A/MART-1.Indeed, the immunogenicity of the Melan-A antigen in melanoma has beenstrongly suggested by the analysis of several active (8, 9) and passive(10-15) immunotherapy protocols targeting this antigen.

The identification of additional melanoma antigens with a documentedimmunogenic potential remains a major issue to address for cancerimmunotherapy, especially for melanoma.

SUMMARY OF THE INVENTION

One object of the invention is a melanoma antigen peptide comprising theamino acids motif:

TX₂NDECWPX₉ (SEQ ID NO: 2)

wherein X₂ is leucine, methionine, valine, isoleucine or glutamine andX₉ is alanine, valine or leucine,

or

RX₂PPKPPLX₉ (SEQ ID NO: 3)

wherein X₂ is cysteine, leucine, methionine, valine, isoleucine orglutamine and X₉ is alanine, valine or leucine.

Another object of the invention is an expression vector comprising anucleic acid sequence encoding the melanoma antigen peptide of theinvention.

Another object of the invention is a host cell comprising saidexpression vector.

Another object of the invention is an antibody or fragment thereof thatbinds to MELOE-1 or MELOE-2.

Another object of the invention is a MHC/peptide multimer comprising amelanoma antigen peptide of the invention.

Another object of the invention is an immunising composition comprising

(a) at least one melanoma antigen peptide of the invention or

(b) at least one expression vector of the invention, or

(c) at least one host cell of the invention, or

(d) at least one antibody of the invention, or

(e) at least one nucleic acid sequence that encodes at least onemelanoma antigen peptide of the invention.

Another object of the invention is a T lymphocyte that recognizesspecifically a melanoma antigen peptide of the invention.

Another object of the invention is a composition for adoptive therapycomprising said T lymphocytes.

Another object of the invention is a method for producing said Tlymphocytes comprising the steps of:

(a) stimulating PBMCs or TIL obtained from a subject with at least onemelanoma antigen peptide of the invention,

(b) enriching the population of T lymphocytes specific for the melanomaantigen peptide(s) used in (a),

(c) optionally cloning said population of T lymphocytes specific for themelanoma antigen peptide(s) used in (a).

Another object of the invention is said immunising composition forpreventing or treating melanoma in a subject in need thereof.

Another object of the invention is an in vitro method for diagnosing amelanoma in a subject in need thereof, comprising detecting theexpression of at least one of:

-   -   MELOE mRNA (SEQ ID NO: 1),    -   MELOE-1 polypeptide (SEQ ID NO: 4),    -   MELOE-2 polypeptide (SEQ ID NO: 5),

in a sample obtained from said subject.

Another object of the invention is a method for monitoring a melanoma ina subject in need thereof, comprising determining the frequency of Tlymphocytes that recognize specifically a melanoma antigen peptide ofthe invention

DETAILED DESCRIPTION OF THE INVENTION

Definitions

As used herein, the term “peptide” refers to an amino acid sequencehaving less than 50 amino acids. As used herein, the term “peptide”encompasses amino acid sequences having less than 50 amino acids, lessthan 40 amino acids, less than 30 amino acids, less than 25 amino acids,less than 20 amino acids, less than 15 amino acids or less than 10 aminoacids.

As used herein, the term “antibody” refers to a protein capable ofspecifically binding an antigen, typically and preferably by binding anepitope or antigenic determinant or said antigen. The term “antibody”also includes recombinant proteins comprising the binding domains, aswell as variants and fragments of antibodies. Examples of fragments ofantibodies include Fv, Fab, Fab′, F(ab′)2, dsFv, scFv, sc(Fv)2,diabodies and multispecific antibodies formed from antibody fragments.

“Function-conservative variants” as used herein refer to those in whicha given amino acid residue in a protein or enzyme has been changed(inserted, deleted or substituted) without altering the overallconformation and function of the polypeptide. Such variants includeprotein having amino acid alterations such as deletions, insertionsand/or substitutions. A “deletion” refers to the absence of one or moreamino acids in the protein. An “insertion” refers to the addition of oneor more of amino acids in the protein. A “substitution” refers to thereplacement of one or more amino acids by another amino acid residue inthe protein. Typically, a given amino acid is replaced by an amino acidwith one having similar properties (such as, for example, polarity,hydrogen bonding potential, acidic, basic, hydrophobic, aromatic, andthe like). Amino acids other than those indicated as conserved maydiffer in a protein so that the percent protein or amino acid sequencesimilarity between any two proteins of similar function may vary and maybe, for example, from 70% to 99% as determined according to an alignmentscheme such as by the Cluster Method, wherein similarity is based on theMEGALIGN algorithm. A “function-conservative variant” also includes apolypeptide which has at least 60% amino acid identity as determined byBLAST or FASTA algorithms, preferably at least 75%, more preferably atleast 85%, still preferably at least 90%, and even more preferably atleast 95%, and which has the same or substantially similar properties orfunctions as the native or parent protein to which it is compared. Twoamino acid sequences are “substantially homologous” or “substantiallysimilar” when greater than 80%, preferably greater than 85%, preferablygreater than 90% of the amino acids are identical, or greater than about90%, preferably greater than 95%, are similar (functionally identical)over the whole length of the shorter sequence. Preferably, the similaror homologous sequences are identified by alignment using, for example,the GCG (Genetics Computer Group, Program Manual for the GCG Package,Version 7, Madison, Wis.) pileup program, or any of sequence comparisonalgorithms such as BLAST, FASTA, etc.

The term “Major Histocompatibility Complex” (MHC) is a genericdesignation meant to encompass the histo-compatibility antigen systemsdescribed in different species including the human leucocyte antigens(HLA).

The term “melanoma” as used herein includes, but is not limited to,melanomas, metastatic melanomas, melanomas derived from eithermelanocytes or melanocytes related nevus cells, melanocarcinomas,melanoepitheliomas, melanosarcomas, melanoma in situ, superficialspreading melanoma, nodular melanoma, lentigo maligna melanoma, acrallentiginous melanoma, ocular melanoma invasive melanoma or familialatypical mole and melanoma (FAM-M) syndrome. Such melanomas in mammalsmay be caused by, chromosomal abnormalities, degenerative growth anddevelopmental disorders, mitogenic agents, ultraviolet radiation (UV),viral infections, inappropriate tissue expression of a gene, alterationsin expression of a gene, or carcinogenic agents.

The term “treating” a disorder or a condition refers to reversing,alleviating or inhibiting the process of one or more symptoms of suchdisorder or condition. The term “preventing” a disorder or conditionrefers to preventing one or more symptoms of such disorder or condition.

The term “diagnosing” as used herein refers to the process ofidentifying a medical condition or disease. As used herein, the term“marker” or “biomarker” refers to a molecule used as a target foranalysing subject's biological samples.

A “biological sample” as used herein refers to a variety of sample typesobtained from a subject and can be used in a diagnostic or monitoringassay. Biological samples include but are not limited to blood, serum,plasma, ascitis, effusions, solid tissue samples such as a biopsyspecimen or tissue cultures or cells derived there from, and the progenythereof, clinical samples, cells in culture, cell supernatants, celllysates. For example, biological samples include cells obtained from atissue sample collected from a subject suspected of having a melanoma.

As used herein, the term “subject” denotes a mammal, such as a rodent, afeline, a canine, and a primate. Preferably a subject according to theinvention is a human.

A “therapeutically effective amount” as used herein is intended for aminimal amount of active agent which is necessary to impart therapeuticbenefit to a subject. For example, a “therapeutically effective amountof the active agent” to a subject is an amount of the active agent thatinduces, ameliorates or causes an improvement in the pathologicalsymptoms, disease progression, or physical conditions associated withthe disease affecting the subject.

The term “adjuvant” as used herein refers to a compound or a mixturethat may be non-immunogenic when administered in the host alone, butthat augments the host's immune response to an antigen when administeredconjointly with that antigen.

The Invention

In order to identify new melanoma antigens, the inventors studied TILpopulations that had been infused to melanoma patients in an adjuvantsetting, after invaded lymph node excision, between 1994 and 2002, andwho are still relapse-free (16). The authors previously showed acorrelation between the infusion of melanoma specific TIL and relapseprevention (17). More recently, they showed a correlation between theinfusion of Melan-A/MART-1 specific TIL and relapse prevention of HLA-A2treated patient (14).

Nonetheless, in a number of TIL populations infused to relapse-freepatients, a significant fraction of tumor-specific TIL remains ofunknown specificity. In order to fully characterize these tumor specificTIL and to look for new tumor antigen(s) involved in relapse prevention,the inventors used a TIL population infused to M170 patient in 1998, whois still relapse-free today (18). This HLA-A2 TIL population contained asignificant fraction of melanoma reactive TIL, among which Melan-A/A2specific lymphocytes and lymphocytes of unknown specificity werepresent.

The study of these lymphocytes of unknown specificity led the inventorsto identify a new mRNA sequence (SEQ ID NO: 1) named meloe(melanoma-overexpressed) that is overexpressed in melanoma, and thatencodes polypeptides recognized by autologous TIL in the HLA-A2 context.

The invention provides a nucleic acid sequence SEQ ID NO: 1, whichencodes two novel melanoma antigens peptides recognized by T cells:MELOE-1 and MELOE-2.

MELOE-1 is encoded by the open reading frame 1230-1370 bp of SEQ ID NO:1 and MELOE-2 is encoded by the open reading frame 285-404 bp of SEQ IDNO: 1.

The inventors identified two specific peptide motifs: TLNDECWPA inMELOE-1 and RCPPKPPLA in MELOE-2, said motifs being capable of bindingto HLA-A2 molecule and inducing a T cell response.

Concerning the peptide motifs capable of binding to HLA-A2, it is knownin the art that the 2^(nd) amino acid from the N-terminus can beleucine, methionine, valine, isoleucine or glutamine and the amino acidat C-terminus can be leucine or valine (39,40).

Therefore, one object of the invention is a melanoma antigen peptidecomprising the amino acids motif:

(a) TX₂NDECWPX₉ (SEQ ID NO: 2)

wherein X₂ is leucine, methionine, valine, isoleucine or glutamine andX₉ is alanine, valine or leucine,

or

(b) RX₂PPKPPLX₉ (SEQ ID NO: 3)

wherein X₂ is cysteine, leucine, methionine, valine, isoleucine orglutamine and X₉ is alanine, valine or leucine.

In one embodiment of the invention, by “melanoma antigen peptide” ismeant a peptide capable of binding to HLA molecule and causing acellular or humoral response in a subject.

In a first embodiment of the invention, said melanoma antigen peptidemay comprise a specific motif such that the polypeptide binds an HLAmolecule and induces a CTL response.

In a second embodiment of the invention, said melanoma antigen peptidemay comprise a specific motif such that the polypeptide binds an HLAmolecule and induces a helper T cell response.

In one embodiment of the invention, said melanoma antigen peptides asdescribed here above are HLA-A2 restricted.

In one embodiment of the invention, said melanoma antigen peptide is anamino acid sequence of less than 50 amino acids long that comprises theamino acid motif SEQ ID NO: 2 or SEQ ID NO: 3 as defined here above.

In another embodiment of the invention, said melanoma antigen peptide isan amino acid sequence of less than 45 amino acids long that comprisesthe amino acid motif SEQ ID NO: 2 or SEQ ID NO: 3 as defined here above.

In another embodiment of the invention, said melanoma antigen peptide isan amino acid sequence of less than 40 amino acids long that comprisesthe amino acid motif SEQ ID NO: 2 or SEQ ID NO: 3 as defined here above.

In another embodiment of the invention, said melanoma antigen peptide isan amino acid sequence of less than 30 amino acids long that comprisesthe amino acid motif SEQ ID NO: 2 or SEQ ID NO: 3 as defined here above.

In another embodiment of the invention, said melanoma antigen peptide isan amino acid sequence of less than 20 amino acids long that comprisesthe amino acid motif SEQ ID NO: 2 or SEQ ID NO: 3 as defined here above.

In another embodiment of the invention, said melanoma antigen peptide isan amino acid sequence of less than 15 amino acids long that comprisesthe amino acid motif SEQ ID NO: 2 or SEQ ID NO: 3 as defined here above.

In another embodiment of the invention, said melanoma antigen peptide isan amino acid sequence of 9, 10 or 11 amino acids long that comprisesthe amino acid motif SEQ ID NO: 2 or SEQ ID NO: 3 as defined here above.

In one embodiment of the invention, said melanoma antigen peptide isselected in the group consisting of MELOE-1 having the sequence SEQ IDNO: 4 and MELOE-2 having the sequence SEQ ID NO: 5.

SEQ ID NO: 4 MSCVGYPDEATSREQFLPSEGAACPPWHPSERISSTLNDECWPASL SEQ ID NO: 5MSENAGGAVARTATAFCALVSPTPQPRCPPKPPLAALCQ

In another embodiment of the invention, said melanoma antigen peptide isselected in the group consisting of MELOE-1 peptides having the sequenceSEQ ID NO: 6 to SEQ ID NO: 20 and MELOE-2 peptides having the sequenceSEQ ID NO: 21 to SEQ ID NO: 38.

MELOE-1 SEQ ID NO 6 X₂ = L X₉ = A TLNDECWPA MELOE-1 SEQ ID NO 7 X₂ = MX₉ = A TMNDECWPA MELOE-1 SEQ ID NO 8 X₂ = V X₉ = A TVNDECWPAMELOE-1 SEQ ID NO 9 X₂ = I X₉ = A TINDECWPA MELOE-1 SEQ ID NO 10 X₂ = QX₉ = A TQNDECWPA MELOE-1 SEQ ID NO 11 X₂ = L X₉ = V TLNDECWPVMELOE-1 SEQ ID NO 12 X₂ = M X₉ = V TMNDECWPV MELOE-1 SEQ ID NO 13 X₂ = VX₉ = V TVNDECWPV MELOE-1 SEQ ID NO 14 X₂ = I X₉ = V TINDECWPVMELOE-1 SEQ ID NO 15 X₂ = Q X₉ = V TQNDECWPV MELOE-1 SEQ ID NO 16 X₂ = LX₉ = L TLNDECWPL MELOE-1 SEQ ID NO 17 X₂ = M X₉ = L TMNDECWPLMELOE-1 SEQ ID NO 18 X₂ = V X₉ = L TVNDECWPL MELOE-1 SEQ ID NO 19 X₂ = IX₉ = L TINDECWPL MELOE-1 SEQ ID NO 20 X₂ = Q X₉ = L TQNDECWPLMELOE-2 SEQ ID NO 21 X₂ = C X₉ = A RCPPKPPLA MELOE-2 SEQ ID NO 22 X₂ = LX₉ = A RLPPKPPLA MELOE-2 SEQ ID NO 23 X₂ = M X₉ = A RMPPKPPLAMELOE-2 SEQ ID NO 24 X₂ = V X₉ = A RVPPKPPLA MELOE-2 SEQ ID NO 25 X₂ = IX₉ = A RIPPKPPLA MELOE-2 SEQ ID NO 26 X₂ = Q X₉ = A RQPPKPPLAMELOE-2 SEQ ID NO 27 X₂ = C X₉ = V RCPPKPPLV MELOE-2 SEQ ID NO 28 X₂ = LX₉ = V RLPPKPPLV MELOE-2 SEQ ID NO 29 X₂ = M X₉ = V RMPPKPPLVMELOE-2 SEQ ID NO 30 X₂ = V X₉ = V RVPPKPPLV MELOE-2 SEQ ID NO 31 X₂ = IX₉ = V RIPPKPPLV MELOE-2 SEQ ID NO 32 X₂ = Q X₉ = V RQPPKPPLVMELOE-2 SEQ ID NO 33 X₂ = C X₉ = L RCPPKPPLL MELOE-2 SEQ ID NO 34 X₂ = LX₉ = L RLPPKPPLL MELOE-2 SEQ ID NO 35 X₂ = M X₉ = L RMPPKPPLLMELOE-2 SEQ ID NO 36 X₂ = V X₉ = L RVPPKPPLL MELOE-2 SEQ ID NO 37 X₂ = IX₉ = L RIPPKPPLL MELOE-2 SEQ ID NO 38 X₂ = Q X₉ = L RQPPKPPLL

The invention also encompasses peptides that are function-conservativevariants of melanoma antigen peptides comprising SEQ ID NO: 2 or SEQ IDNO: 3 as described here above.

Typically, the invention encompasses peptides substantially identical tomelanoma antigen peptides comprising SEQ ID NO: 2 or SEQ ID NO: 3 inwhich one or more residues have been conservatively substituted with afunctionally similar residue and which displays the functional aspectsof the melanoma antigen peptides comprising SEQ ID NO: 2 and SEQ ID NO:3 as described here above, i.e. being still able to bind to an HLAmolecule in substantially the same way as a peptide consisting of thegiven amino acid sequence.

Examples of conservative substitutions include the substitution of onenon-polar (hydrophobic) residue such as isoleucine, valine, leucine ormethionine for another, the substitution of one polar (hydrophilic)residue for another such as between arginine and lysine, betweenglutamine and asparagine, between glycine and serine, the substitutionof one basic residue such as lysine, arginine or histidine for another,or the substitution of one acidic residue, such as aspartic acid orglutamic acid or another.

The term “conservative substitution” also includes the use of achemically derivatized residue in place of a non-derivatized residue.“Chemical derivative” refers to a subject peptide having one or moreresidues chemically derivatized by reaction of a functional side group.Examples of such derivatized molecules include for example, thosemolecules in which free amino groups have been derivatized to form aminehydrochlorides, p-toluene sulfonyl groups, carbobenzoxy groups,t-butyloxycarbonyl groups, chloroacetyl groups or formyl groups. Freecarboxyl groups may be derivatized to form salts, methyl and ethylesters or other types of esters or hydrazides. Free hydroxyl groups maybe derivatized to form O-acyl or O-alkyl derivatives. The imidazolenitrogen of histidine may be derivatized to form N-im-benzylhistidine.Chemical derivatives also include peptides which contain one or morenaturally-occurring amino acid derivatives of the twenty standard aminoacids. For examples: 4-hydroxyproline may be substituted for proline;5-hydroxylysine may be substituted for lysine; 3-methylhistidine may besubstituted for histidine; homoserine may be substituted for serine; andornithine may be substituted for lysine.

In one embodiment of the invention, the melanoma antigen peptideconsists essentially of an amino acid sequence according to SEQ ID NO: 6to 38 or a variant thereof.

According to the invention, “consisting essentially of shall mean that apeptide according to the present invention, in addition to the sequenceaccording to any of SEQ ID No. 6 to SEQ ID No. 38 or a variant thereof,contains additional N- and/or C-terminally located stretches of aminoacids that are not necessarily forming part of the peptide thatfunctions as core sequence of the peptide comprising the binding motifand as an immunogenic epitope.

According to the invention, the melanoma antigen peptides of theinvention can be obtained by synthesizing the peptides according to themethod for peptide synthesis known in the art.

Another object of the invention is an expression vector comprising anucleic acid sequence encoding an amino sequence comprising SEQ ID NO: 2or SEQ ID NO: 3 as described here above.

In one embodiment of the invention, said expression vector comprises thenucleic acid sequence corresponding to the open reading frame 1230-1370bp of SEQ ID NO: 1.

In another embodiment of the invention, said expression vector comprisesthe nucleic acid sequence corresponding to the open reading frame285-404 bp of SEQ ID NO: 1.

In another embodiment of the invention, said expression vector comprisesthe nucleic acid sequence corresponding to the open reading frame1230-1370 bp of SEQ ID NO: 1 combined to the nucleic acid sequencecorresponding to the open reading frame 285-404 bp of SEQ ID NO: 1.

In another embodiment of the invention, said expression vector comprisesa nucleic acid sequence encoding SEQ ID NO: 4 or SEQ ID NO: 5.

In another embodiment of the invention, said expression vector comprisesa nucleic acid sequence encoding a polypeptide comprising at least oneamino acid sequence selected from SEQ ID NO: 6 to SEQ ID NO: 38.

According to the invention, expression vectors suitable for use in theinvention may comprise at least one expression control elementoperationally linked to the nucleic acid sequence. The expressioncontrol elements are inserted in the vector to control and regulate theexpression of the nucleic acid sequence. Examples of expression controlelements include, but are not limited to, lac system, operator andpromoter regions of phage lambda, yeast promoters and promoters derivedfrom polyoma, adenovirus, retrovirus, lentivirus or SV40. Additionalpreferred or required operational elements include, but are not limitedto, leader sequence, termination codons, polyadenylation signals and anyother sequences necessary or preferred for the appropriate transcriptionand subsequent translation of the nucleic acid sequence in the hostsystem. It will be understood by one skilled in the art that the correctcombination of required or preferred expression control elements willdepend on the host system chosen. It will further be understood that theexpression vector should contain additional elements necessary for thetransfer and subsequent replication of the expression vector containingthe nucleic acid sequence in the host system. Examples of such elementsinclude, but are not limited to, origins of replication and selectablemarkers. It will further be understood by one skilled in the art thatsuch vectors are easily constructed using conventional methods orcommercially available.

Another object of the invention is a host cell comprising an expressionvector as described here above.

According to the invention, examples of host cells that may be used areeukaryote cells, such as animal, plant, insect and yeast cells andprokaryotes cells, such as E. coli. The means by which the vectorcarrying the gene may be introduced into the cells include, but are notlimited to, microinjection, electroporation, transduction, ortransfection using DEAE-dextran, lipofection, calcium phosphate or otherprocedures known to one skilled in the art.

In a preferred embodiment, eukaryotic expression vectors that functionin eukaryotic cells are used. Examples of such vectors include, but arenot limited to, viral vectors such as retrovirus, adenovirus,adeno-associated virus, herpes virus, vaccinia virus, poxvirus,poliovirus; lentivirus, bacterial expression vectors, plasmids, such aspcDNA3 or the baculovirus transfer vectors. Preferred eukaryotic celllines include, but are not limited to, COS cells, CHO cells, HeLa cells,NIH/3T3 cells, 293 cells (ATCC #CRL1573), T2 cells, dendritic cells, ormonocytes.

In one embodiment of the invention, said antibody or fragment thereofbinds to MELOE-1 (SEQ ID NO: 4).

In another embodiment of the invention, said antibody or fragmentthereof binds MELOE-2 (SEQ ID NO: 5).

In one embodiment of the invention, said antibody is monoclonal. Inanother embodiment of the invention, said antibody is polyclonal.

Such antibodies may be easily prepared, for example, according to themethod described in “Antibodies: A laboratory manual”, Lane H. D. et al.eds, Cold Spring Harbor Laboratory Press, New York, 1989 or AntibodyEngineering: Methods and Protocols, 2003, Benny K. Lo.

Another object of the invention is a MHC/peptide multimer comprising amelanoma antigen peptide (SEQ ID NO: 6 to 38) as described here above.According to the invention, said MHC/peptide multimer include, but arenot limited to, a MHC/peptide dimer, trimer, tetramer or pentamer.

In one embodiment of the invention, said MHC/peptide multimer is aHLA-A2/peptide multimer.

Methods for obtaining MHC/peptide tetramers are described in WO96/26962and WO01/18053, which are incorporated by reference.

In one embodiment of the invention, said MHC/peptide multimer can beused to visualise T cell populations that are specific for the complexHLA-A2/melanoma antigen peptide as described here above.

In another embodiment of the invention, said MHC/peptide multimer can beused for the detection and/or isolation by screening (in flow cytometryor by immunomagnetic screening) of T cell population that are specificfor a complex HLA/melanoma antigen peptide as described here above.

In another embodiment of the invention, said HLA-A2/peptide multimer canbe used for the detection and/or isolation by screening (in flowcytometry or by immunomagnetic screening) of T cell population that arespecific for a complex HLA-A2/melanoma antigen peptide as described hereabove.

Another object of the invention is beads coated with MHC/peptidemultimers as described here above.

Another object of the invention is an immunising composition comprising

-   -   (a) at least one melanoma antigen peptide as described here        above or    -   (b) at least one expression vector as described here above, or    -   (c) at least one host cell as described here above, or    -   (d) at least one antibody as described here above, or    -   (e) at least one nucleic acid sequence that encodes at least one        melanoma antigen peptide as described here above.

In one embodiment, said immunising composition comprises the melanomaantigen peptide MELOE-1 having the sequence SEQ ID NO: 4 or the melanomaantigen peptide MELOE-2 having the sequence SEQ ID NO: 5.

In another embodiment of the invention, said immunising compositioncomprises at least one melanoma antigen peptide selected in the groupconsisting of SEQ ID NO: 6 to SEQ ID NO: 38.

The prophylactic administration of the immunising composition of theinvention should serve to prevent or attenuate melanoma in a mammal. Ina preferred embodiment mammals, preferably human, at high risk formelanoma are prophylactically treated with the immunising composition ofthe invention. Examples of such mammals include, but are not limited to,humans with a family history of melanoma, humans with a history ofatypical moles, humans with a history of FAM-M syndrome or humansafflicted with melanoma previously resected and therefore at risk forreoccurrence.

When provided therapeutically, the immunising composition of theinvention is provided to enhance the patient's own immune response tothe melanoma antigen present on the melanoma or metastatic melanoma.

In one embodiment of the invention, the peptides of the invention may beconjugated with lipoprotein or administered in liposomal form or withadjuvant.

In one embodiment, said immunising composition is a pharmaceuticalcomposition.

In such embodiment, said immunising composition, for human use,comprises at least one melanoma antigen peptide as described here aboveor at least one antibody as described here above, together with one ormore pharmaceutically acceptable carriers and, optionally, othertherapeutic ingredients. The carrier(s) must be “acceptable” in thesense of being compatible with the other ingredients of the compositionand not deleterious to the recipient thereof. The immunisingcompositions may conveniently be presented in unit dosage form and maybe prepared by any method well-known in the pharmaceutical art.

Immunising compositions suitable for intravenous, intradermal,intramuscular, subcutaneous, or intraperitoneal administrationconveniently comprise sterile aqueous solutions of the active agent withsolutions which are preferably isotonic with the blood of the recipient.Such compositions may be conveniently prepared by dissolving solidactive ingredient in water containing physiologically compatiblesubstances such as sodium chloride (e.g. 0.1-2.0M), glycine, and thelike, and having a buffered pH compatible with physiological conditionsto produce an aqueous solution, and rendering said solution sterile.These may be present in unit or multi-dose containers, for example,sealed ampoules or vials.

The immunising compositions of the invention may incorporate astabilizer. Illustrative stabilizers are polyethylene glycol, proteins,saccharides, amino acids, inorganic acids, and organic acids which maybe used either on their own or as admixtures. These stabilizers arepreferably incorporated in an amount of 0.11-10,000 parts by weight perpart by weight of active agent. If two or more stabilizers are to beused, their total amount is preferably within the range specified above.These stabilizers are used in aqueous solutions at the appropriateconcentration and pH. The specific osmotic pressure of such aqueoussolutions is generally in the range of 0.1-3.0 osmoles, preferably inthe range of 0.8-1.2. The pH of the aqueous solution is adjusted to bewithin the range of 5.0-9.0, preferably within the range of 6-8.

Additional pharmaceutical methods may be employed to control theduration of action. Controlled release preparations may be achievedthrough the use of polymer to complex or absorb the peptides of theinvention. The controlled delivery may be exercised by selectingappropriate macromolecules (for example polyester, polyamino acids,polyvinyl, pyrrolidone, ethylenevinylacetate, methylcellulose,carboxymethylcellulose, or protamine sulfate) and the concentration ofmacromolecules as well as the methods of incorporation in order tocontrol release. Another possible method to control the duration ofaction by controlled-release preparations is to incorporate the melanomaantigen peptides of the invention into particles of a polymeric materialsuch as polyesters, polyamino acids, hydrogels, poly(lactic acid) orethylene vinylaceiate copolymers. Alternatively, instead ofincorporating these agents into polymeric particles, it is possible toentrap these materials in microcapsules prepared, for example, bycoacervation techniques or by interfacial polymerization, for example,hydroxy-methylcellulose or gelatin-microcapsules andpoly(methylmethacylate) microcapsules, respectively, or in colloidaldrug delivery systems, for example, liposomes, albumin microspheres,microemulsions, nanoparticles, and nanocapsules or in macroemulsions.

When oral preparations are desired, the compositions may be combinedwith typical carriers, such as lactose, sucrose, starch, talc magnesiumstearate, crystalline cellulose, methyl cellulose, carboxymethylcellulose, glycerin, sodium alginate or gum arabic among others.

Immunisation of a subject with the immunising composition of theinvention can be conducted by conventional methods, for example, in thepresence of conventional adjuvants. Examples of conventional adjuvantinclude, but are not limited to, metal salts, oil in water emulsions,Toll like receptors agonists, saponins, lipid A, alkyl glucosaminidephosphate, Freund's adjuvant, keyhole limpet haemocyanin (KLH), mannan,BCG, alum, cytokines such as IL-1, IL-2, macrophage colony stimulatingfactor, and tumor necrosis factor; and (6) other substances that act asimmunostimulating agents such as muramyl peptides or bacterial cell wallcomponents, toxins, toxoids and TLR ligands.

The immunising composition can be administered by any route appropriatefor antibody production and/or T cell activation such as intravenous,intraperitoneal, intramuscular, subcutaneous, and the like. Theimmunising composition may be administered once or at periodic intervalsuntil a significant titer of anti-MELOE-1 or MELOE-2 immune cells oranti-MELOE-1 or MELOE-2 antibody is produced. The presence ofanti-MELOE-1 or MELOE-2 immune cells may be assessed by measuring thefrequency of precursor CTL (cytoxic T-lymphocytes) against the melanomaantigen peptides of the invention prior to and after immunization byspecific tetramer labelling (13) or by a CTL precursor analysis assay(41). The antibody may be detected in the serum using an immunoassay.

Antibodies directed to the melanoma antigens of the invention can alsobe used directly as anti-melanoma agents. To prepare antibodies, a hostanimal may be immunized using the MELOE-1 (SEQ ID NO: 4) or MELOE-2 (SEQID NO: 4) protein or others melanoma antigen peptides as described hereabove. The host serum or plasma is collected following an appropriatetime to provide a composition comprising antibodies reactive to saidmelanoma antigen peptides. The gamma globulin fraction or the IgGantibodies can be obtained, for example, by use of saturated ammoniumsulfate or DEAE Sephadex, or other techniques known to those skilled inthe art. The antibodies are substantially free of many of the adverseside effects which may be associated with other anti-cancer agents suchas chemotherapy.

The immunising composition of the invention comprising antibodies asdescribed here above can be made even more compatible with the hostsystem by minimizing potential adverse immune system responses. This isaccomplished by removing all or a portion of the Fc portion of a foreignspecies antibody or using an antibody of the same species as the hostsubject, for example, the use of antibodies from human/human hybridomas.Humanized antibodies (i.e., nonimmunogenic in a human) may be produced,for example, by replacing an immunogenic portion of an antibody with acorresponding, but nonimmunogenic portion (i.e., chimeric antibodies).Such chimeric antibodies may contain the reactive or antigen bindingportion of an antibody from one species and the Fc portion of anantibody (nonimmunogenic) from a different species. Examples of chimericantibodies, include but are not limited to, nonhuman mammal-humanchimeras, rodent-human chimeras, murine-human and rat-human chimeras.

Methods for obtaining said antibodies, chimeric antibodies and humanizedchimeric antibodies are well-known in the art.

The immunising composition comprising the antibodies of the inventioncan also be used as a means of enhancing the immune response. Theantibodies can be administered in amounts similar to those used forother therapeutic administrations of antibody. For example, pooled gammaglobulin is administered at a range of about 1 mg to about 100 mg persubject. Thus, antibodies reactive with the melanoma antigen peptides ofthe invention can be passively administered alone or in conjunction withother anti-cancer therapies to a mammal afflicted with melanoma.Examples of anti-cancer therapies include, but are not limited to,chemotherapy, radiation therapy, adoptive immunotherapy therapy withTIL.

The antibodies or chimeric antibodies described herein may also becoupled to toxin molecules, radioisotopes and drugs by conventionalmethods. Examples of toxins to which the antibodies may be coupled toinclude, but are not limited to, ricin or diphtheria toxin. Examples ofdrugs or chemotherapeutic agents include, but are not limited to,cyclophosphamide or doxorubicin. Examples of radioisotopes, include, butare not limited to, ¹³¹I. Antibodies covalently conjugated to theaforementioned agents can be used in cancer immunotherapy for treatingmelanoma.

If the subject to be immunized is already afflicted with melanoma ormetastatic melanoma, the immunising composition of the invention can beadministered in conjunction with other therapeutic treatments. Examplesof other therapeutic treatments includes, but are not limited to,adoptive T cell immunotherapy, coadministration of cytokines or othertherapeutic drugs for melanoma.

In one embodiment of the invention, said immunising compositioncomprising

-   -   (a) at least one melanoma antigen peptide as described here        above or    -   (b) at least one expression vector as described here above, or    -   (c) at least one host cell as described here above, or    -   (d) at least one antibody as described here above, or    -   (e) at least one nucleic acid encoding at least one melanoma        antigen peptide of the invention,

may further comprise

(a′) at least one MART-1 or Melan-A peptide as described in EP1630229,or

(b′) at least one expression vector comprising a nucleic acid encoding aMART-1 peptide, or

(c′) at least one host cell comprising the expression vector of (b′), or

(d′) at least one antibody or fragment thereof that recognizesspecifically a MART-1 peptide, or

(e′) at least one nucleic acid encoding a MART-1 peptide.

Examples of MART-1 peptides include, but are not limited to, AAGIGILTV(SEQ ID NO: 39), EAAGIGILTV (SEQ ID NO: 40), AAGIGILTVI (SEQ ID NO: 41),ELAGIGILTV (SEQ ID NO: 42).

The dose of melanoma antigen peptides of the invention or MART-1peptides to be administered to a subject may be adjusted as appropriatedepending on, for example, the disease to be treated, the age and thebody weight of said subject. Ranges of melanoma antigen peptides of theinvention or MART-1 peptides that may be administered are about 0.001 toabout 100 mg per subject, preferred doses are about 0.01 to about 10 mgper subject.

The immunising composition of the invention may be evaluated first inanimal models, initially rodents, and in nonhuman primates and finallyin humans. The safety of the immunization procedures is determined bylooking for the effect of immunization on the general health of theimmunized animal (weight change, fever, appetite behavior etc.) andlooking for pathological changes on autopsies. After initial testing inanimals, melanoma cancer patients can be tested. Conventional methodswould be used to evaluate the immune response of the patient todetermine the efficiency of the immunising composition.

Another object of the invention is an antigen presenting cell comprisinga complex HLA antigen and a melanoma antigen peptide of the invention.

In one embodiment of the invention, said complex HLA antigen is a HLA-A2antigen.

In one embodiment of the invention, said antigen presenting cell isderived from the subject to be treated.

The term “antigen presenting cell” (APCs) refers to any cell thatexpresses an HLA antigen capable of presenting the melanoma antigenpeptide of the invention on its surface. Dendritic cells, which arereported to have an especially high antigen-presenting ability, arepreferred. In another embodiment, artificial APCs may also be used suchmammalian cells (fibroblast, endothelial cells, keratinocytes), insectcells, or cell lines.

In order to prepare such APCs of the invention, cells having anantigen-presenting ability are isolated from the subject to be treated,and pulsed ex vivo with at least one melanoma antigen peptide of theinvention to form a complex with the HLA-A2 antigen.

In case dendritic cells are used, the APC of the invention can beprepared as follows. Lymphocytes are isolated from peripheral blood ofthe subject to be treated by Ficoll method; adherent cells are separatedfrom non-adherent cells; the adherent cells are then cultured in thepresence of GM-CSF and IL-4 to induce dendritic cells; and the dendriticcells are pulsed by culturing with at least one melanoma antigen peptideof the invention to obtain the APCs of the invention. The dendriticcells should be exposed to the melanoma antigen peptide for sufficienttime to allow the antigens to be internalized and presented on thedendritic cells surface. The resulting dendritic cells can then bere-administrated to the subject to be treated. Such methods aredescribed in WO93/208185 and EP0563485, which are incorporated byreference.

Another object of the invention is a composition for activeimmunotherapy comprising antigen presenting cells comprising a complexHLA antigen and a melanoma antigen peptide of the invention.

In one embodiment of the invention, said antigen presenting cellscomprise a complex HLA-A2 antigen and a melanoma antigen peptide of theinvention.

Said APCs may be preferably contained in physiological saline, phosphatebuffered saline (PBS), culture medium, or the like. Administration maybe achieved, for example, intravenously, hypodermically, orintradermally.

By returning the above described composition for active immunotherapyinto the subject's body, specific CTL may be efficiently induced in thepatient who is positive for meloe, and thereby tumor can be treated.

Another object of the invention is a T lymphocyte that recognizesspecifically the melanoma antigen peptide of the invention.

In one embodiment of the invention, said T lymphocyte is a cytotoxic Tlymphocyte.

In another embodiment of the invention, said T lymphocyte is HLA-A2restricted.

In another embodiment of the invention, said T lymphocyte is a T cellclone.

In another embodiment, said T lymphocyte is a genetically modified Tlymphocyte that expresses a TCR that recognizes specifically themelanoma antigen peptide of the invention.

Another object of the invention is a composition for adoptive therapycomprising said T lymphocytes as described here above that recognizesspecifically the melanoma antigen peptide of the invention.

In the case of melanoma, it has been observed that an adoptiveimmunotherapy wherein intratumoral T cell infiltrate taken from thesubject to be treated are cultured ex vivo in large quantities, and thenreturned into the patient achieves a therapeutic gain.

It is preferred that the T cells are contained in physiological saline,phosphate buffered saline (PBS), culture medium, or the like in order totheir stable maintain. Administration may be achieved, for example,intravenously or intra-tumoraly. By returning the T cells thatrecognizes specifically the melanoma antigen peptide of the inventioninto the subject's body, the toxicity of said T cells on tumor cells isenhanced in the patient who is positive for meloe. The tumor cells aredestroyed and thereby the treatment of tumor is achieved.

Examples of where T-lymphocytes can be isolated, include but are notlimited to, peripheral blood cells lymphocytes (PBL), lymph nodes, ortumor infiltrating lymphocytes (TIL).

Such lymphocytes can be isolated from tumor or peripheral blood of theindividual to be treated by methods known in the art and cultured invitro. Lymphocytes are cultured in media such as RPMI or RPMI 1640 for2-5 weeks, preferably for 2-3 weeks. Viability is assessed by trypanblue dye exclusion assay. The lymphocytes are exposed to the melanomaantigen peptide of the invention for all of the culture duration.

In a preferred embodiment the lymphocytes are exposed to the melanomaantigen peptide of the invention at a concentration of about 1 to about10 micrograms(μg)/ml per 10⁷ cells for all the duration of lymphocyteculture. Cytokines may be added to the lymphocyte culture such as IL-2.

The melanoma antigen peptide of the invention may be added to theculture in presence of antigen presenting cells such as dendritic cellsor allogeneic irradiated melanoma cell line cells.

After being sensitized to the peptide, the T-lymphocytes areadministered to the subject in need of such treatment.

Examples of how these sensitized T-cells can be administered to themammal include but are not limited to, intravenously, intraperitoneallyor intralesionally. Parameters that may be assessed to determine theefficacy of these sensitized T-lymphocytes include, but are not limitedto, production of immune cells in the subject being treated or tumorregression. Conventional methods are used to assess these parameters.Such treatment can be given in conjunction with cytokines or genemodified cells (Rosenberg, S. A. et al. (1992) Human Gene Therapy, 3:75-90; Rosenberg, S. A. et al. (1992) Human Gene Therapy, 3: 57-73).

Another object of the invention is a composition for adoptive therapycomprising HLA-A2 restricted T lymphocytes that recognizes specificallythe melanoma antigen peptide of the invention, which further comprisesHLA-A2 restricted T lymphocytes that recognize specifically aMART-1/Melan-A peptide.

HLA-A2 restricted T lymphocytes that recognize specifically aMART-1/Melan-A peptide are known in the art and were described inVignard et al., 2005.

Another object of the invention is a method for producing T lymphocytesthat recognize specifically a melanoma antigen peptide of the invention,said method comprising the steps of:

(a) stimulating PBMCs or tumor infiltrating lymphocytes (TIL) obtainedfrom a subject with at least one melanoma antigen peptide of theinvention,

(b) enriching the population of T lymphocytes specific for the melanomaantigen peptide(s) used in (a),

(c) optionally cloning said population of T lymphocytes specific for themelanoma antigen peptide(s) used in (a).

Enrichment and/or cloning may be carried out by using an MHC/peptidemultimer as described here above. Cloning may also be carried out byconventional methods.

In one embodiment of the invention, the T lymphocytes that recognizespecifically a melanoma antigen peptide of the invention are HLA-A2restricted. In such embodiment, enrichment and/or cloning may be carriedout by using an HLA-A2/peptide multimer as described here above.

Stimulation of PBMCs may be carried out with at least one melanomaantigen peptide of the invention alone, or presented by an antigenpresenting cell such as dendritic cells or allogeneic irradiatedmelanoma cell line cells. Typically, cytokines such as IL-2 may also beadded to the culture.

Another object of the invention is a composition for adoptive therapythat comprises lymphocytes that recognizes specifically the melanomaantigen peptide of the invention for preventing or treating melanoma ina subject in need thereof, wherein said T lymphocytes are to bere-administrated to the subject.

In one embodiment, said lymphocytes that recognize specifically themelanoma antigen peptide of the invention are HLA-A2 restricted.

Another object of the invention is an immunising composition comprising

-   -   (a) at least one melanoma antigen peptide as described here        above or    -   (b) an expression vector comprising a nucleic acid sequence        encoding a melanoma antigen peptide defined in (a) as described        here above, or    -   (c) a host cell comprising an expression vector defined in (b)        as described here above, or    -   (d) an antibody that recognizes specifically a melanoma antigen        peptide defined in (a) as described here above, or    -   (e) at least one nucleic acid encoding at least one melanoma        antigen peptide of the invention,

for preventing or treating melanoma in a subject in need thereof.

Another object of the invention is a composition for immunotherapy thatcomprises antigen presenting cells comprising a HLA molecule and amelanoma antigen peptide of the invention.

In one embodiment, said complex HLA/peptide is a complex HLA-A2/melanomaantigen peptide of the invention.

The invention also relates to a method for treating melanoma in asubject in need thereof, comprising administering a therapeuticallyeffective amount of

-   -   (a) at least one melanoma antigen peptide as described here        above or    -   (b) an expression vector as described here above, or    -   (c) a host cell as described here above, or    -   (d) an antibody as described here above, or    -   (e) at least one nucleic acid encoding at least one melanoma        antigen peptide of the invention.

The invention also relates to a method for treating melanoma in asubject in need thereof, comprising administering a therapeuticallyeffective amount of T lymphocytes that recognizes specifically themelanoma antigen peptide of the invention. In one embodiment, said Tlymphocytes are HLA-A2 restricted.

The invention also relates to a method for treating melanoma in asubject in need thereof, comprising administering a therapeuticallyeffective amount of antigen presenting cells comprising a complex HLAantigen and a melanoma antigen peptide of the invention. In oneembodiment, said complex HLA/peptide is a complex HLA-A2/melanomaantigen peptide of the invention.

The inventors observed that meloe cDNA expression level was higher inmelanomas than in melanocytes and that meloe expression was very low inother tumor cell lines such as breast or lung tumor cell lines.

Therefore, one object of the invention is meloe as a biomarker ofmelanoma.

Another object of the invention is an in vitro method for diagnosing amelanoma in a subject in need thereof, comprising detecting theexpression of at least one of:

-   -   meloe mRNA (SEQ ID NO: 1),    -   MELOE-1 peptide (SEQ ID NO: 4),    -   MELOE-2 peptide (SEQ ID NO: 5),

in a sample obtained from said subject.

Examples of methods for determining the transcription level of meloecDNA include, but are not limited to Northern blotting, RNaseprotection, polymerase chain reaction (PCR), quantitative PCR, real-timePCR.

Meloe mRNA and MELOE-1 and MELOE-2 proteins are present in melanomacells. It is therefore an aspect of the invention to provide meloenucleic acid probes to be utilized in detecting meloe RNA or MELOE-1 orMELOE-2 proteins or alterations in the level of meloe mRNA or MELOE-1 orMELOE-2 proteins in biological sample isolated from a subject afflictedwith melanomas. By alterations in the level of meloe mRNA or MELOE-1 orMELOE-2 proteins, we mean an increase or decrease in the level of a RNAor MELOE-1 or MELOE-2 proteins relative to a control sample or theappearance or disappearance of the meloe mRNA or MELOE-1 or MELOE-2proteins relative to a control sample. Detection in the alterations ofmeloe mRNA or MELOE-1 or MELOE-2 proteins will allow for diagnosis orthe assessment of the diseased state. Therefore, alterations in thelevel of meloe mRNA or MELOE-1 or MELOE-2 proteins may be predictive ofthe prognosis for the afflicted mammal.

In another embodiment, the meloe nucleic acid of this invention can beused in in situ hybridization on mammalian tissues to determine theprecise site or subcellular site of expression of the meloe gene withina tissue. A preferred method of labelling the meloe nucleic acidsequence is synthesizing a ³⁵S-labeled RNA probe by in vitrotranscription utilizing SP6 polymerase. Conventional methods forpreparation of tissues for in situ, synthesis of probes and detection ofsignal are known in the art. The probe is then contacted with mammaliantissue sections and in situ analyses performed by conventional methods.Examples of tissues that can be used include, but are not limited to,mammalian embryos, adult mammalian tissues, such as skin, lymph nodesand retina, biopsy specimens, pathology specimens and necropsyspecimens. In a preferred embodiment, meloe in situ probes may be usedto evaluate meloe RNA expression in diseased tissue for invasive earlymelanoma to characterize radial and vertical growth phases of themelanoma lesion and assess the margins of the disease within the tissue.

In another embodiment of the invention, the antibodies of this inventionmay be used in immunoassays to detect the MELOE-1 or MELOE-2 protein inbiological samples. In this method, the antibodies of the invention arecontacted with a biological sample and the formation of a complexbetween the melanoma antigen peptide and antibody is detected.Immunoassays of the present invention may be radioimmunoassay, Westernblot assay, immunofluorescent assay, enzyme immunoassay,chemiluminescent assay, immunohistochemical assay and the like. Standardtechniques for ELISA are known in the art. Such assays may be direct,indirect, competitive, or noncompetitive immunoassays as described inthe art. Biological samples appropriate for such detection assaysinclude mammalian tissues, melanoma and melanocyte cell lines, skin,retina, lymph nodes, pathology specimens, necropsy specimens, and biopsyspecimens. Proteins may be isolated from biological samples byconventional methods.

The antibodies of this invention can therefore be used in immunoassaysto detect MELOE-1 or MELOE-2 proteins or alteration in the level ofexpression of these proteins in biological samples isolated from mammalsafflicted with a disease or disorder. Examples of biological samplesinclude, but are not limited to, mammalian tissues, biopsy tissuesamples, melanoma and lymph node biopsy samples, pathology and tissuesamples. Examples of diseases that can be assessed by theseimmunoassays, include, but are not limited to, melanomas and tissueswhich are secondary sites for melanoma metastasis. By alteration inlevel of expression, we mean an increase or decrease of the MELOE-1 orMELOE-2 protein or portions thereof relative to a control sample. Theantibodies of this invention can therefore be used in an immunoassay todiagnose, assess or prognoses a mammal afflicted with melanoma.

In another embodiment of the invention, rabbit antisera containingantibodies which specifically recognize the melanoma antigen peptides ofthe invention may be used to detect said peptides in Western BlotAnalysis. Using conventional methods, rabbits may be immunized with atleast one melanoma antigen peptides of the invention conjugated tocarriers. Preferably about 0.1 to about 10 mg of antigen in adjuvant maybe used, most preferably about 1 mg of antigen in adjuvant may be used.The animal receives similar booster doses and antisera titer is assessedby ELISA assay. Satisfactory levels of antisera are obtained when theanti-peptide antibody titer reaches a plateau. This antibody can be usedin the standard immunoassays described above.

Another object of the invention is a method for monitoring a melanoma ina subject in need thereof, comprising determining the frequency of Tlymphocytes that recognize specifically a melanoma antigen peptide ofthe invention.

In one embodiment of the invention, said T lymphocytes are HLA-A2restricted.

In one embodiment of the invention, the frequency of T lymphocytes thatrecognize specifically a melanoma antigen peptide of the invention maybe determined by using an MHC/peptide multimer as described here above.

According to the invention, an increase in the frequency of Tlymphocytes that recognize specifically a melanoma antigen peptide ofthe invention correlates with relapse prevention.

Another object of the invention is a kit comprising:

-   -   an antibody that recognizes specifically MELOE-1 or MELOE-2        and/or    -   primers or probes for meloe mRNA detection, and/or    -   an MHC/peptide multimer comprising a melanoma antigen peptide of        the invention.

In one embodiment, said kit further comprises a solid support, whereinsaid solid support is selected from the group consisting of wells ofreaction trays, test tubes, polystyrene beads, strips, membranes andmicroparticles.

In another embodiment, said kit further comprises a label, wherein saidlabel is selected in the group consisting of enzymes, radioisotopes,fluorescent compounds and chemiluminescent compounds.

The following examples are given for the purpose of illustrating variousembodiments of the invention.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. T cell clone selection and characterization. (A) % of TNFproducing T cells and of HLA-A2/Melan-A_(A27L) tetramer positive T cellsin M170 TIL population in response to the autologous melanoma cell line.10⁵ TIL and 2.10⁵ melanoma cells were incubated for 5 hours, in thepresence of Brefeldin A, stained with HLA-A2/Melan-A_(A27L) tetramer,fixed, and stained with anti-TNF antibody in a permeabilization buffer.10⁴ T cells were then analyzed by flow cytometry. (B and C) TNFsecretion by M170.48 T cell clone (B) and M170.51 CTL clone (C) inresponse to the autologous melanoma cell line. 10⁴ CTL were added to3.10⁴ M170 melanoma cells, in presence of blocking antibodies directedagainst class I, A2 and B/C HLA, diluted to 1/50 (black bars), 1/500(hatched bars) and 1/5000 white bars. CTL clonereactivity was assessedby a TNF release assay.

FIG. 2. (A and B) TNF response of M170.48 CTL clone (A) and M170.51 CTLclone (C) to HLA-A*0201 tumor cell lines. M6 cell line, HLA-A2 negative,was used as a negative control. (C) IFN-γ response of M170.48 andM170.51 CTL clones to HLA-A*0201 melanocytes. M170 cell line was addedas a positive control.

FIG. 3. Characterization of the cDNA coding for the recognized antigen.(A and B) M170.48 (A) and M170.51 (B) TNF responses to COS-7 cells (E/Tratio 1/3) transfected with indicated plasmids. T cell clones were added2 days after the transfection and the CTL clone reactivity was assessedby a TNF release assay. (C) Comparison of the nucleotide sequences ofmeloe and BC008026 cDNAs and localization of this sequence on the HDAC4gene. Indicated nucleotides correspond to SNPs between the meloesequence isolated from M134 and A498 tumor cell lines and the meloesequence isolated from M117 and SW480 cell lines, and the BC008026 cDNAsequence.

FIG. 4. Characterization of meloe derived peptides recognized by M170.48and M170.51 T cell clones. (A) Structure of meloe cDNA. Black boxescorrespond to ORFs tested for recognition by the CTL clone. (B) M170.48and M170.51 TNF responses to COS-7 cells (E/T ratio 1/3) transfectedwith indicated plasmids. T cell clone was added 2 days after thetransfection and the CTL clone reactivity was assessed by a TNF releaseassay. (C) Amino acid sequences of the ORF 1230-1370 and the ORF 285-404of meloe isolated from M134 cDNA library. (D and E) Cytotoxicity ofM170.48 CTL clone (D) and M170.51 CTL clone(E) against peptide-pulsed T2cells. Target cells were chromium labeled for 60 min and incubated for30 min with a range of the indicated peptides. T cell clones were addedat an E/T ratio of 10/1 and chromium release was then measured after a 4h incubation period.

FIG. 5. Preferential expression of meloe cDNA in melanoma cell linesmeasured by qPCR, and impact of meloe expression on specific CTL cloneactivation. (A) Four melanoma, one breast cancer, two renal carcinomaand one lung cancer cell lines were tested by qPCR for the expression ofmeloe. RPLPO and β2 microglobulin gene expression were used as internalcontrols. The relative expression of meloe was calculated afternormalization on the efficiency of PCR reaction and the mean expressionof these two house-keeping genes, reported to its normalized expressionin melanocytes. (B) TNF secretion by the M170.48-CTL clone in responseto HLA-A2 tumor cell lines non transfected (white bars), or transfectedwith meloe (hatched bars) or meloe-1 (black bars) expression plasmids.Tumor cells were transiently transfected with 100 ng of each plasmid,with a lipofectamine reagent kit. 10⁴ CTLs were added to 3.10⁴ targetcells, and the CTL clone reactivity was assessed by a TNF release assay.(C) meloe relative expression measured by qPCR in 16 human healthytissues.

FIG. 6. Detection of MELOE-1/A2 and MELOE-2 specific CTLs in TIL infusedto relapse-free melanoma patients and analysis of the repertoirediversity of MELOE-1 specific TIL. (A) HLA-A2 TIL populations labeledwith the A2/MELOE-1₃₆₋₄₄ tetramer. Upper panel: TIL infused torelapse-free patients and lower panel: TIL infused to patients whorelapsed. TIL were coincubated with MELOE-1 tetramer and anti-CD8 mAb.Values indicate the % of tetramer positive cells among CD8⁻ TIL. (B)Labeling of A2/MELOE-2₂₇₋₃₅ specific T cells in M278 TIL population. TILwere co-labeled with CD8 antibody, A2/MELOE-1₃₆₋₄₄ and A2/MELOE-2₂₇₋₃₅tetramers. Values indicate the fraction of positive T cells among CD8positive TIL. (C) Repertoire diversity of multimer-sorted populationswas evaluated by labeling with 25 anti-Vβ mAbs. Inserts illustrate thepurity of each sorted TIL population, assessed by MELOE-1 specifictetramer labeling.

FIG. 7. Reactivity of MELOE-1/A2 and MELOE-2 specific TIL against HLA-A2tumor cell lines

(A) Lysis of M170 melanoma cell line (black symbols) and of 1355 lungcarcinoma cell line (open symbols) by M170.48 CTL clone and MELOE-1specific TIL populations. ⁵¹Cr-labeled tumor cells were co-cultured withT cells at various E/T ratios. Chromium release in the supernatants wasmeasured after a 4 h incubation period. (B) Cytokine production byM170.48 CTL clone and MELOE-1 specific TIL populations in response toM170 melanoma cells. Effector and target cells were incubated at a 1/2ratio in the presence of Brefeldin A, stained with anti-TNF antibody(white bars), anti-IFN-γ antibody (hatched bars) or anti-IL2 antibody(black bars), and 10⁴ T cells were analyzed by flow cytometry. (C)Production of TNF-α, IFN-γ, GM-CSF and IL-2 by the MELOE-2 specific Tcell clone (M170.51) and the MELOE-2 sorted specific population(s-M278), in response to two HLA-A2 positive melanoma cell lines and aHLA-A2 negative melanoma cell line.

FIG. 8. Frequencies of MELOE-1₃₆₋₄₄ and Melan-A_(A27L) specific T cellsamong PBMC from HLA-A2 healthy donors and melanoma patients. About 10⁸PBMC were sorted with PE-conjugated tetramers according a methodrecently described (42). The entire stained sample was then collected onan Facs Canto (BD Immunocytometry Systems) and analyzed with FlowJowsoftware. The percentage of tetramer-positive cells among CD8⁺lymphocytes was estimated from the number of tetramer-positive cells inthe enriched fraction and from the fraction of CD8⁺ cells in total PBMC.On healthy donors, the frequencies of Melan-A_(A27L)/A2 specificlymphocytes were also estimated, as an internal control of the sortingmethod.

FIG. 9. Phenotypic analysis of ex-vivo circulating A2/MELOE-1 specific Tcells in healthy donors and melanoma patients. After sorting withPE-conjugated tetramers, the lymphocyte preparations were stained with acocktail of Pacific blue-conjugated exclusion markers (CD14, CD16,CD19), CD8^(Amc)Y, CD45RA^(PE-Cy7), CD27^(APC-H7), CD28^(PerCP-Cy5.5),and CD62-L^(FITC), and immediately analyzed by flow cytometry. (A and B)Pattern of expression of CD45RA/CD62L (left) or CD28/CD27 (right) gatedon CD8/MELOE-1 double positive T cells, from healthy donors (A) andmelanoma patients (B). Numbers indicate the fraction of healthy donorsor melanoma patients with the corresponding phenotype.

FIG. 10. Cytotoxicity of MELOE-1 specific CTL clones againstpeptide-pulsed T2 cells. Target cells were chromium labeled for 60 minand incubated for 30 min with a range of the MELOE-1₃₆₋₄₄ peptide. Tcell clones derived from healthy donors (A), melanoma patients PBMC (B)and TIL (C) were added at an E/T ratio of 10/1 and chromium release wasthen measured after a 4 h incubation period. (D) Mean EC50 of theMELOE-1 specific T cell clones in response to the MELOE-1₃₆₋₄₄ peptide,according to their origin. EC50 were compared using statistical analysisdone with InStat 2.01. Data were analyzed using Kruskal-Walliscomparison test. *p=0.019, considered significant.

FIG. 11. Lysis of M170 (HLA-A2 positive, upper panel) and M6 (HLA-A2negative, lower panel) melanoma cell lines by MELOE-1 specific T cellclones derived from healthy donors, melanoma patients PBMC and TIL.⁵¹Cr-labeled tumor cells were co-cultured with T cells at 10/1 and 1/1E/T ratios. Chromium release in the supernatants was measured after a 4h incubation period.

EXAMPLES

Experimental Procedures

Material and Methods

Cell Lines and TIL Cultures

T cell populations were expanded from cryopreserved samples of TIL(derived from tumor invaded lymph nodes) infused to melanoma patientsincluded in a phase I/II protocol. This clinical trial aimed atcomparing the survival of stage III melanoma patients randomly treatedby IL-2 alone or TIL+IL-2, in an adjuvant setting (16). TIL samples wereexpanded according a procedure previously described (32, 33). TILcontaining tumor specific T cells were cloned by limiting dilution (34)and tumor specific T cell clones were amplified as previously described(33). Melanoma cells lines and colorectal carcinoma cell line C4-A wereestablished respectively in the Unit of cellular therapy and in ourlaboratory. Mouse fibrosarcoma WEHI 164 clone 13 and COS-7 cells wereobtained from T. Boon (LICR, Brussels, Belgium). Ovary carcinoma celllines (OVCAR-3, 0114) and renal carcinoma cell line A498 were gift fromC. Saï (INSERM U892, Nantes, France). Colorectal carcinoma cell lines(CaCo-2, Sw480, Sw707, LS174T), renal carcinoma cell line HTC116, breastcarcinoma cell line 734-B, were gifts from M. Grégoire (INSERM U601,Nantes, France), S. Chouaib (INSERM U487, Villejuif, France) and D.Jäger (Klinik and Poliklinik für Onkologie, Zürich, Germany). Breastcancer cell line MCF-7 was obtained from the ATCC. Normal melanocytes(98M09 and 01M08), were gifts from M. Regnier (L′Oréal Laboratory,Paris, France). EBV-B cell lines were gifts from H. Vié (INSERM U601,Nantes, France).

Functional Analysis of T Cells

Cytotoxic activity of T cells was measured in a standard 4-h assayagainst ⁵¹Cr-labeled cells (peptide-loaded T2 cells or tumor cell lines)(23). Measurement of TNF produced by T cells in response to tumor cellsor transfected COS-7 cells (19) was performed as previously described,using WEHI 164 clone 13 cells (35). mAb against HLA class I (cloneW6.32), HLA-B/C (clone B1.23.2), HLA-A2 (clone BB7.2) added to culturesin some experiments, were produced in our laboratory from hybridomasobtained from the ATCC for W6.32 and BB7.2 antibodies and from F.Lemonier (Pasteur Institute, France) for B1.23.2 antibody. Intracellularstaining of cytokines was performed as previously described onstimulated T cells (36). For intracytoplasmic cytokine staining, after a6 h-stimulation period with melanoma cells at an E:T ration of 1:2, inpresence of brefeldin A at 10 μg/mL (Sigma, St Louis Mo., USA), T cellswere labeled with an APC-coupled anti-CD8 antibody (BD Biosciences,France), and fixed for 10 min at room temperature in PBS 4%paraformaldehyde (SIGMA). Fixed lymphocytes were stained for cytokineproduction using anti-TNF-α, anti-IFN-γ, anti-GM-CSF and anti-IL2specific antibodies (BD Biosciences, France), as previously described.After staining, cells were resuspended in PBS and analyzed on a LSR flowcytometer using Cell Quest software.

cDNA Library and ORFs Constructs

The M134 cDNA library have been inserted in pcDNA3.1 as describedpreviously (20) and recombinant plasmids were electroporated intoEscherichia coli XL1 (Stratagene). For screening, 800 pools of 100ampicillin-resistant bacteria were constituted. Plasmid DNA wasextracted from each pool with the QIAprep Spin Miniprep kit (QIAGEN).The positive plasmid was sequenced by the DNA Sequencing Facility of theIFR 26 (Nantes, France). The various ORFs from meloe sequence weregenerated by PCR (see Table I for primers). Oligonucleotides weredesigned with EcoRI and XhoI adaptators for subcloning in pcDNA3, andwith a Kozak sequence (gccaccATG) for upper primer, and a stop codon forlower primer.

Synthetic Peptides

Peptides were purchased from Eurogentec (Angers, France). Purity (>70%or >90% for tetramer production) was controlled by reversed-phasehigh-performance liquid chromatography. Peptides were lyophilized,dissolved in DMSO at 10 mg/mL and stored at −20° C.

Real-Time PCR

Total RNA was extracted from tumor cell lines, melanocytes and B-EBVcell lines by Trizol reagent (Invitrogen, Cergy Pontoise, France). RNAfrom healthy tissues were purchased from Clontech (France). Quality ofRNA samples was controlled using an Agilent bioanalyzer and all thesamples exhibited a RIN>7. Retrotranscription was performed using 1 μgof total RNA, random hexamers and SuperScript II reverse transcriptase(Invitrogen). Relative quantification of meloe, RPLPO andβ2-microglobulin expression was carried out using Brilliant SYBR GreenQPCR in the Mx4000 (Stratagene Europe, Amsterdam, The Netherlands). 10ng of cDNA from samples were added to SYBR green master mix (Stratagenefor Mx4000) with forward and reverse primers (Table I) at 200 nM in afinal volume of 25 μL. Thermal cycling was one step at 95° C. for 10min, followed by 40 cycles at 95° C. for 30 s, 63° C. for 1 min and 72°C. for 1 min. The efficiency of PCR reaction was determined with seriesof 10-fold diluted cDNA from M170, performed in parallel to plot thestandard curves for meloe, RPLPO and β2-microglobulin. Duplicatedilution series were included as standards in each run. Averagethreshold cycle (CT) values from duplicate PCR reactions were normalizedto average CT values for two housekeeping genes (β2-microglobulin andRPLPO) from the same cDNA preparations. The relative expression ratio ofa target gene was calculated based on the PCR efficiency (E) and the CTdeviation between a given cell line (x) and a reference cell line(calibrator), expressed in comparison with the mean of the housekeepinggenes (37).

Ratio=(Etarget)^(ΔCTtarget(calibrator-x))/mean((Ehousekeeping)^(ΔCThousekeeping(calibrator-x)))

Tetramer and Vβ Labeling

HLA-A*0201/MELOE-1 and HLA-A*0201/Melan-A α3-mutated monomers weregenerated by the recombinant protein facility (INSERM U892, Nantes,France), as previously described (38). TIL populations and M170.48 Tcell clone were coincubated for 1 h at 4° C. in the dark with MELOE-1tetramer (10 μg/mL) and CD8 mAb (5.1 μg/mL) and 10⁴ events were analyzedon a FACSCalibur. A panel of 25 anti-Vβ mAbs (Vβ1, −2, −3, −4, −5.1,−5.2, −5.3, −6, −7, −7.2, −8, −9, −11, −12, −13.1, −13.2, −13.6, −14,−16, −17, −18, −20, −21.3, −22 and −23) was used to analyse thediversity of sorted TIL populations (Immunotech Beckman-Coulter,Marseille, France).

Immunomagnetic Cell Sorting and Expansion of T Cell Sorted Populations

HLA-A*0201/MELOE-1 monomers (20 μg/mL) were incubated for 1 h at roomtemperature with 6.7.10⁶ streptavidin-coated beads (Dynabeads M-280streptavidin, DYNAL, Compiegne, France) and washed in PBS-0.1% BSA.5.10⁶ TIL were rotated for 4 hours at 4° C. with monomer-coated beads(23, 38). After ten washes, bead coated cells were expanded using apolyclonal T cell stimulation protocol (33).

Frequency and Status of MELOE-1₃₆₋₄₄ Specific T Cells in HLA-A*0201Healthy Donor and Melanoma Patient PBMC.

10⁸ PBMC were collected from HLA-A2 healthy donors (EFS, Nantes, France)or from HLA-A2 melanoma patients (gift from D. Schadendorf, Universityof Essen, Germany and B. Dreno, Nantes hospital, France). Labeling withPE-conjugated tetramers and sorting were performed according to a methodrecently described (42). PE-conjugated MELOE-1 tetramer was added at aconcentration of 50 μg/mL, and the cells were incubated at 4° C. for 1hr, followed by two washes in 15 ml of ice-cold buffer (PBS+0.1% BSA).The tetramer-stained cells were then resuspended in a volume of 0.9 mLof buffer, mixed with 0.1 mL of anti-PE antibody conjugated magneticmicrobeads (Miltenyi Biotech, France) and incubated at 4° C. for 30 min,followed by two washes with 10 mL of sorter buffer. The cells were thenresuspended in 3 mL of buffer and passed over a magnetized LS column(Miltenyi Biotech, France). The column was washed with 3 mL of bufferthree times and then removed from the magnetic field. The bound cellswere eluted by pushing 5 ml of sorter buffer through the column with aplunger. The resulting enriched fractions were resuspended in 0.5 mL ofsorter buffer, and a small volume was removed for cell counting whilethe rest of the sample was stained with a cocktail offluorochrome-labeled antibodies specific for CD14, CD16, CD19, CD8,CD45RA, CD27, CD28, CD62-L and CD127 (BD Biosciences, France). Theentire stained sample was then collected on an Facs Canto (BDImmunocytometry Systems) and analyzed with FlowJow software. Thepercentage of tetramer-positive cells among CD8⁻ lymphocytes wasestimated from the number of tetramer-positive cells in the enrichedfraction and from the fraction of CD8+ cells in total PBMC.

Functional Analysis of T Cells

The relative avidity of MELOE-1₂₆₋₄₄ specific T cell clones and theiranti-tumor reactivity was measured respectively in a standard 4-h assayagainst ⁵¹Cr-labeled peptide-loaded T2 cells and towards HLA-A2 melanomacell lines. Briefly, target cells (peptide pulsed-T2 or melanoma cells)were incubated with 100 μCi Na₂ ⁵¹CrO₄ (Oris Industrie, Gif-sur-Yvette,France) at 37° C. for 1 h. For peptide recognition assays, T2 cells werepreincubated with a range of MELOE-1₂₆₋₄₄ peptide concentrations for 1 hat 37° C. After the 4 h-co-culture, 25 μL of supernatant were mixed with100 μL of scintillation cocktail (Optiphase Supermix, Wallak, UK) formeasurement of radioactive content. EC50 were compared using statisticalanalysis done with InStat 2.01. Data were analyzed using Kruskal-Walliscomparison test, followed by Dunn'post test. P<0.05 was consideredsignificant.

Results

T Cell Clone Selection and Characterization.

M170 TIL population contained 16% of melanoma-reactive lymphocytes,among them 5% were specific of Melan-A/A2 epitope and 11% were ofunknown specificity (FIG. 1A). This TIL population was then tested forrecognition of a large panel of known antigens transfected into COScells, with the class I HLA molecules of M170 patient (19), and noresponse aside from Melan-A/A2 response could be detected (data notshown), suggesting that this population contained lymphocytes specificfor new tumor antigen(s). In order to characterize them, we derivedtumor reactive CD8⁺ T cell clones by limiting dilution. Eight of theseCTL clones showed reactivity patterns consistent with recognition of (a)new antigen(s) and two of them, hereafter referred to as M170.48 andM170.51, were further characterized in order to determine the HLAcontext restricting its recognition. As illustrated by FIGS. 1B and 1C,the recognition of the autologous melanoma cell line by these two CTLclones occurs in the HLA-A2 context. In order to establish thedistribution of the target antigen, we tested T cell clone reactivitytowards various HLA-A2 tumor cell lines including melanomas, ovariancarcinomas, lung carcinomas, breast carcinomas, renal carcinomas andcolon carcinomas, using a TNF release assay. As shown in FIGS. 2 a and2B, these T cell clones recognized all the HLA-A2 melanoma cell linestested but none of the other HLA-A2 tumor cell types. In addition, thesetwo T cell clones cell clone weakly recognized HLA-A2 melanocytes (FIG.2C). However, this reactivity was much lower than that usually seen withMelan-A/A2 specific T cell clones (data not shown).

Identification of the cDNA Coding for the Antigen.

We screened a cDNA library derived from the M134 melanoma cell line (20)in COS-7 cells cotransfected with pHLA-A*0201 to characterize the Agrecognized by M170.48 and M170.51 T cell clones. Among 800 pools of 100pcDNA tested, the same plasmid pool proved positive for the two CTLclones, and the individual plasmid, coding for the antigen recognized byM170.48 (FIG. 3A) and M170.51 (FIG. 3B) was recovered from it after acloning step. This insert, namely meloe, spanning 2128 bp, was sequencedand was found to contain a poly(A) tail and to be similar to the cloneBC008026 isolated by the NIH MGC consortium (21). After expressionvector cloning, cotransfection of BC008026 cDNA with HLA A*0201 intoCOS-7 also induced the recognition by M170.48 and M170.51 T cell clone(FIGS. 3A and 3B), although these two sequences differed by four SNPs(FIG. 3C). In order to control the impact of these SNPs on therecognition of the two T cell clones, we sequenced the cDNA isolatedfrom recognized (M134, M117) and non recognized cell lines (A498,SW480), and showed that this polymorphism did not affect tumor cell linerecognition. The meloe sequence analysis showed a perfect colinearitywith the genomic DNA, obtained by comparison with the sequence of thehuman genome released by Celera (22), which indicated absence ofsplicing. Finally, this sequence was found to be located in the thirdintron of the HDAC-4 gene (Gene ID 9759), on chromosome 2, in anti-senseorientation compared to the sequence of the HDAC-4 gene.

Identification of the Peptides Recognized by M170.48 and M170.51 T CellClones.

The meloe cDNA does not contain a long ORF, but multiple short ORFs(FIG. 4A). The putative ORF described by the NIH MGC consortium waslocated between 486 and 689 bp (21). We tested this ORF and threeadditional ORFs (black boxes in FIG. 4A), for recognition by M170.48 andM170.51, after transfection into COS-7 cells, with the HLA-A*0201 cDNA(FIG. 4B). The ORFs tested were chosen on the basis of preliminaryresults obtained on PCR fragments of meloe (data not shown).

The ORF 1230-1370 bp encodes the protein bearing the peptide recognizedby the specific M170.48 T cell clone, and the ORF 285-404 bp encodes theprotein bearing the peptide recognized by the specific M170.51 T cellclone (FIG. 4C). These ORFs respectively encode a 46 and a 39amino-acids protein. We tested the recognition of various peptidesderived from these two sequences able to bind to the HLA A*0201, with ahigh stability as predicted by BIMAS analysis(http://www-bimas.cit.nih.gov).

Two nonapeptides, were tested for their recognition by M170.48 T cellclone, after loading on T2 cells. Only the sequence 36-44 <<TLNDECWPA>>was recognized (FIG. 4D and data not shown). We then tested therecognition of two additional peptides derived from this nonapeptide. Weobserved that addition of the serine at the C-terminus (position 45)dramatically decreased the response of our CTL clone (black circles onFIG. 3D) and that deletion of the alanin at the C-terminal end (position44) abrogated the CTL clone response (open circles on FIG. 4D). Inconclusion, the optimal peptide recognized by M170.48 CTL clone appearedto be the nonapeptide 36-44 (TLNDECWPA), with a half maximal lysis of10⁻⁸M (black squares on FIG. 4D).

Several nonapeptides were tested for their recognition by M170.51 T cellclone, after loading on T2 cells. Only the sequence 27-35 <<RCPPKPPLA>>was recognized (FIG. 4E and data not shown). We then tested therecognition of additional peptides derived from this nonapeptide. Weobserved that addition of the alanin at the C-terminus (position 36)dramatically decreased the response of our CTL clone (black circles onFIG. 4E) and that deletion of the arginin at the N-terminal end(position 27) abrogated the CTL clone response (open circles on FIG.4D), even when the leucin was added at the C-terminal end (open squareson FIG. 4D). In conclusion, the optimal peptide recognized by M170.51CTL clone appeared to be the nonapeptide 27-35 (CPPKPPLA), with a halfmaximal lysis of 5.10⁻⁸M (black squares on FIG. 4D).

Meloe is Overexpressed in Melanomas

In order to explain the absence of recognition of tumor cell lines otherthan melanomas, we compared the transcription level of meloe cDNA byqPCR in a panel of HLA-A2 tumor cell lines and melanocytes. The meanlevel of meloe expression in two HLA-A2 melanocytes was used as areference to establish its relative expression in other cell lines. Thisanalysis showed that meloe expression in melanomas was higher than inmelanocytes, with values ranging from 3 to 34 fold higher, whereas thisexpression was significantly lower in other tumor cell lines, withvalues ranging from 5 to 338 fold lower (FIG. 5A). These results showthat this antigen is overexpressed in melanomas and thus proteinsencoded by the ORF 1230-1370 and 285-404 were respectively called“MELOE-1” for “melanoma-overexpressed antigen-1” and MELOE-2 for“melanoma-overexpressed antigen-2”. Furthermore, transfection of meloeor meloe-1 cDNA in HLA-A2 non recognized tumor cell lines induced theirrecognition by M170.48 T cell clone (FIG. 5B), showing that the absenceof recognition of these tumor cell lines was due to the underexpressionof meloe cDNA. Finally, in order to address the question of theexpression of this antigen in healthy tissues, we performed qPCR on apanel of 16 tissues. It appears that the expression of meloe in healthytissues was always lower than in melanocytes. The highest meloeexpression was found in whole and fetal brain but remained respectively1.5 and 1.8 fold below its expression in melanocytes (FIG. 4C). Overall,these results suggest that this antigen could be safely targeted inimmunotherapy protocols in melanoma, provided that its immunogenicitycould be documented.

Presence of MELOE-1 Specific Lymphocytes in TIL Populations Infused toRelapse Free Patients

In order to address the question of the immunogenicity of this newepitope, we used a specific HLA-A2/peptide tetramer to look for thepresence of specific lymphocytes among 30 HLA-A2 TIL populations derivedfrom melanoma invaded lymph nodes. All those TIL populations had beeninfused to melanoma patients in an adjuvant setting, between 1998 and2002. Following this treatment, 21 of these patients relapsed and 9remained relapse-free. Using a specific HLA/peptide tetramer, wedetected the presence of MELOE-1/A2 specific T cells in 5/9 TILpopulations that had been infused to relapse-free patients, withfrequencies ranging from 0.07% to 3.8% among CD8⁺ TIL (FIG. 6A, upperpanel). In contrast, we did not observe the presence of such T cellsamong the TIL infused to the 21 HLA-A2 patients who relapsed. An exampleof 5 out of these 21 negative TIL populations is shown on FIG. 6A, lowerpanel. These results document the existence of a correlation between thepresence of MELOE-1 specific lymphocytes among infused TIL and relapseprevention (p<0.001), and thus suggest the potential immunogenicity ofthis new HLA-A2 melanoma epitope. We also detected MELOE-2 specific Tcells in M278 TIL population, which also contains MELOE-1 specificlymphocytes (FIG. 6B).

Finally, in order to address the question of the diversity and tumorreactivity of the MELOE-1/A2 specific repertoire, specific lymphocyteswere sorted by monomer-based immunomagnetic sorting (23), from the 5positive TIL populations. Inserts on FIG. 6B illustrate the purity ofsorted TIL, assessed by specific tetramer labeling. We also attempted tosort 5 negative TIL populations with monomer-coated beads, but noMELOE-1 specific cells were obtained (data not shown). This last resultformally documented the absence of such cells in those populations, orat least showed that the frequencies of MELOE-1 specific T cells weretoo low to allow their purification by multimer sorting. The diversityof TCR Vβ usage of sorted populations was assessed with a panel of 25anti-Vβ antibodies representing the most frequently expressed Vβ chainswithin a normal repertoire. In M117, M170 and M278 sorted populations, 8or 6 different Vβ chains were significantly expressed (above 1%) byMELOE-1/A2 specific TIL, indicating the presence of a rather polyclonalspecific TCR repertoire (FIG. 6C). TCR diversity of M134 sorted TIL wasmuch lower, with a strong dominance of lymphocytes expressing the Vβ13.1chain (FIG. 6C). This may be related to the low fraction of MELOE-1specific T cells present in this population before sorting (0.3% of CD8⁺TIL, FIG. 6A), probably poorly diverse. Finally, we could not determinethe dominant Vβ chain expressed by TIL sorted from M180, with our panelof antibodies. Therefore, no dominant Vβ usage could be observed withinthese three sorted TIL populations. Finally, in order to support thepotential role of MELOE-1/A2 specific TIL transfer in relapseprevention, we studied the reactivity of sorted TIL populations onHLA-A*0201 melanoma cell lines that spontaneously express the MELOE-1antigen. All sorted T cell lines were lytic against melanoma cell lines(FIG. 7A and data not shown) and produced IFN-γ and TNF upon stimulationby these cells, with levels similar to M170.48 CTL clone, and to a lowerextent IL-2 (FIG. 7B and data not shown). MELOE-2 specific lymmphocytessorted from M278 TIL population were also reactive against HLA-A2melanoma cell lines, as illustrated by cytokine production in responseto a stimulation with M170 HLA-A2 melanoma line (FIG. 7C).

Frequency and Status of MELOE-1₃₆₋₄₄ Specific T Cells in HLA-A2 HealthyDonors and Melanoma Patients PBMC and TIL.

10⁸ PBMC were sorted by tetramer labeling according the method describedby Moon et al. (20). Sorted cells were immediately analyzed bymulticolor staining The frequency of MELOE-1 specific T cells among CD8⁺lymphocytes was deduced from the total number of CD8/tetramer positivesorted cells and the total number of CD8 lymphocytes among PBMC beforesorting. As a control, we performed the same analysis on Melan-Aspecific CD8 T cells. The number of tetramer positive cells recoveredafter the sorting step ranged between 50 and 400 for MELOE-1 specificcells and between 400 and 4000 for Melan-A specific cells. Concerninghealthy donors, the frequency of MELOE-1₂₆₋₄₄ specific T cells amongCD8+ cells ranged from 1.8 10⁻⁶ to 1.8 10⁻⁵, whereas that ofMelan-A_(A27L) specific T cells ranged from 1.8 10⁻⁵ to 2.5 10⁻⁴ (FIG.8, black symbols). The frequencies observed in melanoma patients wererather similar, ranging from 2.2 10⁻⁶ to 1.7 10⁻⁵ for MELOE-1 specific Tcells and from 2.2 10⁻⁵ to 2 10⁻⁴ for Melan-A specific lymphocytes (FIG.8, open symbols). CD8/MELOE-1 specific T cells were then phenotyped forCD45RA, CD62L, CD28 and CD27 by mutiparametric flow cytometry (43, 44).

Circulating A2/MELOE-1 CD8+ T cells detected in 4/6 healthy donorspresented a predominant naive phenotype(CD45RA^(hi)/CD28⁻/CD27⁺/CD62L⁺), (FIG. 9A), while in 2/6 donors,tetramer⁺ cells displayed high proportions (69 and 58%) of memory Tcells (CD45RA^(low)/CD28⁺/CD27⁺/CD62L⁺) (FIG. 9A). Concerning melanomapatients, we also observed these two different phenotypes. Indeed,MELOE-1 specific T cells from 3/6 patients mainly displayed a naivephenotype (FIG. 9B). In contrast, in the remaining 3 patients, 59 to 85%of A2/MELOE-1 specific cells displayed a memory phenotype.

Relative Avidity and Tumor Reactivity of MELOE-1 Specific T Cell Clones

15/18 clonotypes could be further amplified to analyse their avidity forT2 cells loaded with the MELOE-1 specific peptide. As shown on FIG. 4A,the EC50 of the clonotypes derived from healthy donors ranged from 0.5to 10⁻³ μM of MELOE-1₃₆₋₄₄ peptide (FIG. 10A). The avidity of MELOE-1specific clonotypes derived from patient PBMC or TIL was less scattered,with EC50 ranging respectively from 5.10⁻² to 4.10⁻⁴ μM and from 8.10⁻⁴to 8.10⁻⁷ μM (FIGS. 10B and C). Overall, MELOE-1 specific clonotypesderived from melanoma patients appear to have a better avidity towardsMELOE-1₃₆₋₄₄ peptide, than specific clonotypes derived from healthydonors (FIG. 10D), although all express the Vα12.1 chain. Thisdifference was significant (p=0.019) between T cell clones derived fromhealthy donor PBMC and those derived from melanoma TIL.

Tumor reactivity of the different clonotypes was then tested on HLA-A2melanoma cell lines. FIG. 11 illustrates the results obtained on aHLA-A2 positive melanoma cell line (M170: upper panel) and a HLA-A2negative melanoma cell line (M6: lower panel). M171.1 clonotype couldnot be tested for tumor reactivity because it failed to be furtherexpanded. All the clonotypes derived from patients (PBMC and TIL) werecytotoxic towards M170 cell line, whereas only 2 out of the 5 clonotypesderived from healthy donors were lytic against this cell line (HD1.21and HD3.38). These two clonotypes displayed the better EC50 of the 5clonotypes derived from healthy donors: respectively 10⁻² and 10⁻³ μM.

Discussion

The meloe gene is located on the third intron of the histonedeacetylase-4 gene (HDAC-4), and translated in anti-sense orientation incomparison with the HDAC-4 gene (24). There is a perfect colinearitybetween the meloe gene and its corresponding cDNA, showing an absence ofsplicing of this gene. Furthermore, the structure of this 2.1 kb cDNA israther unusual, with multiple short open reading frames, instead of aunique long ORF. Two proteins respectively encoded by the ORF₁₂₃₀₋₁₃₇₀(MELOE-1) and the ORF₂₈₅₋₄₀₄ (MELOE-2) contain peptides that wererecognized by CTL clones derived from melanoma specific TIL, in theHLA-A*0201 context. These CTL clones recognized all the HLA-A*0201melanoma cell lines tested, and to a lower extent HLA-A*0201melanocytes. On the other hand, these T cell clones failed to recognizeany of the other tumor cell lines tested (FIG. 2).

The meloe antigen could be classified into the family of <<melanocyticdifferentiation antigens>>, due to its expression in melanocytes andmelanoma cell lines (25). Nonetheless, it could be also classified intothe family of aberrantly expressed antigens, due to the particularlocation of meloe gene in the third intron of HDAC-4 gene, following theexample of NA17-A antigen, located in an intron of the GnT-V gene (6).Uunlike classical differentiation antigens such as Melan-A or tyrosinase(18, 26, 27), we detected by quantitative PCR a residual expression ofmeloe gene in other cancer cell lines, even if this expression level wastoo low to induce their recognition by our specific CTL clone, asrecently shown for a mouse prostate tumour epitope derived from histoneH4 (28). meloe also differs from a classical differentiation antigen byits overexpression in melanomas, compared to normal melanocytes. Thus,meloe seems to be specifically overexpressed in melanomas and therefore,this new antigen presents both the properties of tissue specificity andoverexpression in cancer. The reasons of its overexpression in melanomascould be due to the regulation of meloe promoter. As described forMAGE-1 gene, a transient and general process of demethylation in tumorscould be followed by a persistent inhibition of remethylation due to thepresence of melanoma specific transcription factors (29). In this way,meloe overexpression in melanomas could be due to the hypomethylation ofits promoter in melanoma cell lines. In another way, melanoma specifictranscription factors, such as MITF which controls the expression of thethree main melanocytic differentiation antigens (30, 31), could alsocontrol the overexpression of meloe in melanomas.

Due to its expression widely shared by melanoma cell lines, and to theexistence of epitopes recognized by melanoma reactive CTL clones in theHLA-A*0201 context, the MELOE-1 and MELOE-2 antigens could be promisingtargets for future immunotherapy protocols of melanoma, provided thattheir usage remains safe in patients and that their immunogenicity couldbe documented.

Our data provides some arguments concerning the safety of immunizationof patients with this antigen or of adoptive transfer with specific CTL.Indeed, the expression level of meloe in healthy tissues is always lowerthan in melanocytes, which are weakly recognized by MELOE-1 and MELOE-2specific CTL clones, to a lower extent than by Melan-A specific CTL(FIGS. 4C, 1C and data not shown). Furthermore, none of the otherHLA-A*0201 cancer cell lines was able to spontaneously induce anyreactivity of the two specific CTL (FIG. 2), even after a 48 h-treatmentwith IFN-γ (data not shown). MELOE-1 specific CTL clone could only beactivated by such tumor cell lines when they were previously transfectedby the meloe cDNA or the cDNA coding for ORF₁₂₃₀₋₁₃₇₀ (FIG. 5B), showingthat the expression level of meloe in other cancer cell lines was toolow to induce the activation of specific lymphocytes. This expressionlevel being similar to that measured in healthy tissues by qPCR (FIG.5C), this suggests that an immunization with this antigen should notinduce deleterious reactions in healthy tissues, although we couldexpect the induction of some vitiligo due to the expression of meloe inmelanocytes.

The second point concerns the immunogenicity of these new antigens andespecially the immunogenicity of the HLA-A*0201 restricted epitopes. Toanswer this main question, we checked the presence of MELOE-1 specificlymphocytes among TIL populations that had been infused to stage IIImelanoma patients after lymph node excision, in an adjuvant setting. Inretrospective studies of this adoptive transfer protocol, we alreadyshowed that the infusion of melanoma specific TIL had a significantimpact on relapse prevention of treated patient (17). More recently wefound that prolonged relapse-free survival of TIL-treated patientscorrelated with the infusion of Melan-A specific lymphocytes (14),although a significant fraction of tumor-specific TIL remains of unknownspecificity in a number of TIL populations infused to relapse-freepatients. We detected a significant fraction of MELOE-1 specificlymphocytes, by tetramer labeling, in 5/9 TIL populations infused toHLA-A2 patients who remained relapse-free since 7 to 13 years, whereasno MELOE-1 specific lymphocytes could be observed in 21 TIL populationsinfused to patients who relapsed. Interestingly, we also found MELOE-2reactive TIL in M170 and M278 TIL populations (data not shown). Thestatistical analysis of these results performed by a Chi² test,documents a correlation between the prevention of relapse of TIL treatedHLA-A2 patients and the presence of MELOE-1 specific CTLs among thoseTIL (p<0.001). This main result provides a strong argument in favor ofthe implication of MELOE-1 antigen in the immunosurveillance of patientstreated by adoptive transfer of TIL. This role in immunosurveillance hasnot been formally elucidated for the majority of melanoma antigensidentified, except for Melan-A/MART-1, which seems clearly involved inclinical responses of melanoma patients treated by immunotherapy (10-12,14, 15). Following the example of Melan-A antigen, a diverse and tumorreactive T cell repertoire is necessary to develop protocols of adoptivetransfer of specific T cells in melanoma patients, and also forvaccination trials. We addressed this last issue by analyzing thediversity and reactivity of MELOE-1 specific repertoire in the 5 TILpopulations that contained MELOE-1 specific T cells. After selection ofMELOE-1 specific T cells from TIL by immunomagnetic sorting withmultimer-coated beads (23), we showed that MELOE-1 specific repertoirewas diverse in 3/5 TIL populations, and much more limited for M134 TILpopulation, which contained a low fraction of MELOE-1 specific T cells(FIGS. 6A and 6B), that could explain this poorer diversity. We couldnot determine the dominant Vβ chain(s) expressed by M180 sorted TIL withour panel of antibodies (FIG. 6B). The repertoire analysis of these TILpopulations did not reveal any recurrence of a particular Vβ usage, aspreviously described for Melan-A specific repertoire (23). These fiveMELOE-1 specific TIL populations were as reactive as the M170.48specific T cell clone against HLA-A2 melanoma cell lines, as shown bylysis (FIG. 7A) and IFN-γ and TNF-β production in response to melanomacells, and, to a lower extent, IL-2 production (FIG. 7B). Furthermore,we recently demonstrated that MELOE-1 specific T cells could be alsoselected and amplified from PBMC of melanoma patients (data not shown),that remains the most convenient source of tumor specific T cells usablefor all HLA-A2 patients enrolled in adoptive transfer protocols.

Using a method recently described of tetramer enrichment (42), we haveenumerated and phenotyped ex-vivo MELOE-1 specific T cells present inHLA-A2 healthy donors and patient peripheral blood. Results show thatMELOE-1 specific T cells are present in each healthy donor and patient,at similar frequencies : around 10⁻⁵ among CD8⁺ cells. This frequency inaround ten times lower than the frequency of Melan-A specific T cells.With the exception of the Melan-A antigen, few studies have establishedex-vivo frequencies of tumor antigen specific T cells in peripheralblood, their low frequencies precluding their direct evaluation.Nonetheless, frequencies of MAGE-3/A1 specific T cells have beenevaluated in healthy donors by a limiting dilution approach and reveal afrequency as low as 2.10⁻⁷ among CD8⁺ T cells. This frequency rangedbetween 7.10⁻⁷ and 3.10⁻³ in patients who were immune responders to avaccination with a recombinant virus encoding the antigen, while thisfrequency remains around 5.10⁻⁷ in non responder patients (45). The onlystudy reporting a high frequency of CTL specific for a tumor specificantigen concerns CTLs specific for NY-ESO-1 antigen (around 2.10⁻⁴ amongCD8⁺), observed in only one melanoma patient (46). Therefore, thepresence of MELOE-1 specific T cells in HLA-A2 melanoma patients, with afrequency around 10⁻⁵ among CD8⁺ lymphocytes supports the development ofimmunotherapy protocols targeting this antigen, and this is strengthenedby a recent study demonstrating the functional relevance of CD8⁺precursor frequency to tumor immunity (47).

REFERENCES

All the cited references are incorporated by reference.

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1. A melanoma antigen peptide comprising the amino acids motif:TX₂NDECWPX₉ (SEQ ID NO: 2)

wherein X₂ is leucine, methionine, valine, isoleucine or glutamine andX₉ is alanine, valine or leucine, or RX₂PPKPPLX₉ (SEQ ID NO: 3)

wherein X₂ is cysteine, leucine, methionine, valine, isoleucine orglutamine and X₉ is alanine, valine or leucine.
 2. A melanoma antigenpeptide according to claim 1, wherein said melanoma antigen peptide isselected in the group consisting of MELOE-1 having the sequence SEQ IDNO: 4 and MELOE-2 having the sequence SEQ ID NO:
 5. 3. A melanomaantigen peptide according to claim 1, wherein said melanoma antigenpeptide is selected in the group consisting of peptides having thesequence SEQ ID NO: 6 to SEQ ID NO:
 38. 4. An expression vectorcomprising a nucleic acid sequence encoding a melanoma antigen peptideaccording to claim
 1. 5. A host cell comprising an expression vectoraccording to claim
 4. 6. An antibody or fragment thereof that binds tothe melanoma antigen peptide according to claim
 2. 7. A MHC/peptidemultimer comprising a melanoma antigen peptide according to claim
 3. 8.An immunising composition comprising (a) at least one melanoma antigenpeptide according to claim 1 or (b) at least one expression vectoraccording to claim 4, or (c) at least one host cell according to claim5, or (d) at least one antibody according to claim 6, or (e) at leastone nucleic acid sequence that encodes at least one melanoma antigenpeptide according to claim
 1. 9. A T lymphocyte that recognizesspecifically a melanoma antigen peptide according to claim
 1. 10. Acomposition for adoptive therapy comprising T lymphocytes according toclaim
 9. 11. A method for producing said T lymphocytes according toclaim 9, said method comprising the steps of: (a) stimulating PBMCs ortumor infiltrating lymphocytes obtained from a subject with at least onemelanoma antigen peptide according to claim 1, (b) enriching thepopulation of T lymphocytes specific for the melanoma antigen peptide(s)used in (a), (c) optionally cloning said population of T lymphocytesspecific for the melanoma antigen peptide(s) used in (a).
 12. A methodfor preventing or treating melanoma in a subject in need thereof,comprising the administration to said subject of the composition foradoptive therapy according to claim 10 wherein said T lymphocytes thatrecognize specifically a melanoma antigen peptide are to bere-administrated to the subject.
 13. A method for preventing or treatingmelanoma in a subject in need thereof, comprising the administration tosaid subject of immunising composition according to claim
 8. 14. An invitro method for diagnosing a melanoma in a subject in need thereof,comprising detecting the expression of at least one of: MELOE mRNA (SEQID NO: 1), MELOE-1 polypeptide (SEQ ID NO: 4), MELOE-2 polypeptide (SEQID NO: 5), in a sample obtained from said subject.
 15. A method formonitoring a melanoma in a subject in need thereof, comprisingdetermining the frequency of T lymphocytes that recognize specifically amelanoma antigen peptide according to claim 1.